Erythropoietin (EPO) is a naturally formed glycoprotein which functions as a colony stimulating factor and serves as the principal factor involved in the regulation of red blood cell synthesis. Erythropoietin acts by stimulating precursor cells in bone marrow causing them to divide and differentiate into mature red blood cells. This process is tightly controlled in the body such that the destruction or removal of red cells from the circulation is matched by the rate of new cell formation. Naturally occurring EPO is a glycoprotein produced in the kidney (Jacobs, et al. Nature 313 (6005), 806–810 (1985).
Erythropoietin has been manufactured using recombinant DNA technology through the cloning of the EPO gene and expression in Chinese hamster ovary cells (Lin, U.S. Pat. No. 5,618,698). The recombinantly produced EPO has been available for some time as an effective therapeutic agent in the treatment of various forms of anemia, including anemia associated with chronic renal failure, zidovidine treated HIV infected patients, and cancer patients on myelosuppressive chemotherapy. The glycoprotein is administered parenterally, either as an intravenous (IV) or subcutaneous (SC) injection in conventional buffered aqueous solutions which contain human serum albumin (HSA) as a carrier. Such formulations are marketed in the United States under the trade names EPOGEN® and PROCRIT®. These products contain erythropoietin in 1 ml single dose, preservative-free or 2 ml multidose preserved vials.
While these formulations have been proven to be highly successful, certain disadvantages are associated with the products. Presently, the period of bioactivity of protein therapeutics such as erythropoietin is limited by short plasma half-lives and the susceptibility to protease degradation. The short half-life of therapeutic proteins such as EPO, four hours, necessitates frequent administration for maximum clinical efficacy. This is disadvantageous for the treatment of chronic conditions and can result in poor patient compliance, and therefore less than optimal outcome. Accordingly, attempts have been made to increase the plasma half-life of EPO.
In recent years, non-antigenic water-soluble polymers, such as polyethylene glycol (PEG) have been used for the covalent modification of polypeptides of therapeutic and diagnostic importance. For example, covalent attachment of PEG to therapeutic polypeptides such as the interleukins (Knauf, M. J. et al., J. Biol. Chem. 1988, 263, 15, 064; Tsutsumi, Y. et al., J. Controlled Release 1995, 33, 447), interferons (Kita, Y. et al., Drug Des Delivery 1990, 6, 157), catalase (Abuchowski, A. et al., J. Biol. Chem. 1977, 252, 3, 582), superoxide dismutase (Beauchamp, C. O. et al., Anal Biochem. 1983, 131, 25), and adenosine deaminase (Chen, R. et al, Biochim, Biophys. Acta 1981, 660, 293), has been reported to extend their half-life in vivo, and/or reduce their immunogenicity and antigenicity.
Derivatized PEG compounds have been previously disclosed (U.S. Pat. No. 5,438,040, Aug. 1, 1995, Conjugation-Stabilized Polypeptide Compositions, Therapeutic Delivery and Diagnostic Formulations Comprising Same, and Method of Making and Using the Same, N. N. Ekwuribe). This approach to post-translational derivatization has also been applied to EPO. For example, WO 94/28024 discloses carbohydrate modified polymer conjugates with erythropoietin activity wherein the PEG is linked via an oxidized carbohydrate. U.S. Pat. No. 4,904,584 discloses polyalkylene oxide conjugation of lysine-depleted polypeptide variants, including EPO. WO 90/12874 describes the preparation of a monomethoxy-PEG-EPO (mPEG-EPO) in which the EPO contains a cysteine residue introduced by genetic engineering to which the specific PEG reagent is covalently attached. Other PEG-EPO compositions are disclosed in EP 605693, U.S. Pat. No. 6,077,939, WO 01/02017 and EP 539167.
Applicant's co-pending application U.S. Ser. No. 09/431,861 discloses the modification of antibodies and antibody fragments with PEG and demonstrates that PEG can increase circulating half-life in mice and primates. Derivatized PEG was used for modification of the Fab fragment of the antibody c7E3. Circulating half-life is increased in direct proportion to the molecular weight of the PEG. As the molecular weight of PEG increases, the ability of the compound to inhibit ADP-induced platelet aggregation in vitro is decreased, while the binding to purified GPIIb/IIIa, as measured by BIAcore, is unaffected. The addition of a fatty acid or a lipid to the PEG (PEG3.4K-DSPE [disteroylphosphatidylethanolamine]) yielded a greater circulating half-life than did PEG5K. While there is a decrease in the in vitro activity of c7E3 Fab′(PEG5k)2 relative to c7E3 Fab, the activity of c7E3 Fab′-(PEG3.4k-DSPE)2 is equivalent to c7E3 Fab.
Applicant's other co-pending application U.S. Ser. No. 60/377,946 discloses methods for modifying EPO in which the EPO is covalently conjugated to a non-antigenic hydrophilic polymer covalently linked to an organic molecule that increases the circulating serum half-life of the composition more than what can be achieved by addition of a hydrophilic polymer alone. The methods include the step of reacting a protein or glycoprotein having erythropoietic activity with a substantially non-antigenic functionalized hydrophilic polymer having a linking group for attaching the polymer to the glycoprotein. Preparation methods include reacting EPO with an activated form of a polyalkylene oxide that will react with a functional group on EPO. This includes activated polyalkylene oxides such as active esters, hydrazide, hydrazine, semicarbazide, thiosemicarbazide maleimide or haloacetyl polyalkylene oxide.
An often limiting aspect of many methods of modifying proteins by conjugation to PEG (“PEGylation”) using purely chemical methods, is the indiscriminate and often incomplete reaction with amine groups which may occur on accessible lysine residues and/or the N-terminal amine of the protein. Other chemical methods require oxidation of the carbohydrate groups as part of the modification strategy likewise leading to incomplete or inconsistent reactions and undefined product compositions. Thus, considering the present options available, a method for modifying EPO in a mild, site-specific manner would be advantageous.
Transglutaminases (TGases) [EC2.3.2.13; protein-glutamine:gamma-glutamyltransferase] are a family of proteins that catalyze the calcium-dependent acyl addition to a primary amine wherein the gamma-carboxamide group of peptide-bound glutamine residue is the acyl donor and the primary amine is the acyl acceptor and amine donor. In nature, TGases crosslink proteins by catalyzing the formation of amide bonds between lysine and glutamine residues on opposing proteins. A well-known example is fibrin cross-linking by the TGase factor XIIIa. This bond is stable and resistant to proteases and thus, TGases are generally used to link structural components of cells. In addition to the above mentioned plasma form, TGases are found in tissues such as liver, skin, and extracellular fluids (Greenberg, C. Set al. FASEB J. 1991, 5, 3071–3077). Prokaryotic forms of TGase are also known (Ando, H. et al. Agric. Biol. Chem 53 (10), 2613–2617, 1989; Washizu, K. et al. Biosci. Biotech. Biochem 58(1), 82–87, 1994). The specificity of TGases is quite pronounced with usually only one, or in some cases two, glutamine residues per protein serving as amine acceptors. TGases from various mammalian tissues and species have been extensively studied (Folk, J. E. and Chung, S. I. Adv. Enzym. Molec. Biol. 1973, 38, 109–191; Folk, J. E. and Finlayson, J. S. Adv. Protein Chem. 1977, 31, 1–133; Folk, J. E & Cole, P. W. Biochim Biophys. Acta 1966, 122, 244–264; Folk, J. E.; Chung, S. I. Methods in Enzymology 1985, 113, 358–375;). Thus, TGases could and have been employed to site-specifically modify glutamine residues on some proteins (U.S. Pat. No. 6,010,871; U.S. Pat. No. 6,331,422; U.S. Pat. No. 6,322,996). 
Despite numerous studies, few details about the determinants of TGase specificity have been elucidated. TGases differ in substrate specificities, and when choosing residues as acyl donors or acceptors, the preference for specific sequence motifs as containing or neighboring the substrate residue has not generally been identified for individual enzymes (Gorman, J. J.; Folk, J. E. J. Biol. Chem. 1981, 256, 2712–2715; Gorman, J. J.; Folk, J. E. J. Biol. Chem. 1980, 255, 419–427). The only definitive rule is that a glutamine residue must be positioned at least three residues from the N-terminus to serve as a substrate for any TGase. In general, glutamine repeats have been shown to enhance the acceptor properties of each glutamine residue in the repeat, and the accessibility of glutamine residues has also been shown to be important in determining their ability to function as TGase substrates (Kahlem, P. et al. Proc. Natl. Acad. Sci. USA 1996, 93, 14580–14585).
Although the site-specific nature of TGase modifications has been known since the 1960s, and industrial uses in the food stabilization are practiced, only recently have uses in therapeutic protein modification begun to be explored. The use of TGases to attach 5 kilodalton or larger polymers containing aliphatic amino groups to protein bound glutamine residues was recently disclosed by Sato, et al in U.S. Pat. No. 6,322,996. This patent also discloses the methods of engineering proteins to contain added N-terminal or C-terminal peptides which are known to be TGase substrates for the purpose of subsequent attachment of large polymers using TGase catalysis. The PEGylation of IL-2 has been accomplished using these methods (Sato, H.; Ikeda, M.; Suzuki, K.; Hirayama, K. Biochemistry 1996, 35, 13072–13080), the cross-linking of IL-2 to various other proteins using bacterial TGase was also demonstrated (Takahara, Y. et al. U.S. Pat. No. 6,010,871), and use of factor XIIIa in the production of a modified fibrin matrix for tissue engineering (U.S. Pat. No. 6,331,422).
The modification or addition of motifs to a naturally occurring molecule carries multiple risks that are well known to those practicing the art of genetic engineering for the purposes of providing manufacturing methods for therapeutic proteins. The most obvious of these effects is the loss or partial loss of biological activity. In other cases, the expression level from constructed expression vectors is unacceptably low when incorporated into mammalian cell lines. The alternate approach of coupling or fusion of a known substrate sequence from a naturally occurring protein substrate may create an antigenic epitope and cause unwanted immune reactions in the subject which ultimately limit the long term efficacy of the therapeutic protein. Furthermore, the modification of proteins using chemical methods that attack the most reactive functional group, lysine, also changes the isoelectric point of the protein and the pKa. Therefore, when the objective is to provide safe and economically produced products, it is important to understand these limitations. The conversion of the amide group of glutamine to an alkylated amine does not change the isoelectric point or charge of that glutamine. Thus, use of an enzymatic process that creates a stable covalent bond while not modifying the electrical charge of the protein would be desirable. Heretofore, EPO has not been considered a natural TGase substrate nor has the re-engineering of the molecule in order to create or eliminate TGase substrate sites in EPO been described.